Molecular Genetic Studies of Thrombosis

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1. Analysis of Genetic Abnormalities for Protein C Deficiency. Makinal use of the polymerase chain reaction (PCR), we analyzed the genes for protein C from Japanese patients with a hereditary protein C deficiency, and identified two molecular abnormalities in two independent probands. One is a 3414T to C mutation in exon VI which converts Tyrl24 (ZAC) to His (CAC) in the second EGF domain. The other is a frameshift deletion of a single G nucleotide in a region of 8854G-8857G in exon IX coding for the carboxy-terminal region, which is predicted to result in an alteration and elongation of the deduced carboxy-terminal amino acid sequence.2. Diagnosis of Familial Protein C Deficiency. Based on a result described above, we have established a direct diagnostic method of familial protein C deficiency in the second case of a frameshift deletion by use of mutacenic primers for PCR to introduce Ava I or Bal I site into the amplified product.3. Polymorphism in the Protein C Gene. We have identified two sequence variations in the protein C gene. The first one is an A-T sequence polymorphism at nucleotide -1476 in the untranslated exon 1, and the second one is a T-G sequence polymorphism in the triplet coding for Ser-99 (TCT - TCG).4. Heparin-Binding Property of Protein C. We found that protein C, and more strongly activated protein C, interacts with heparin, which is enhanced in the presence of calcium ions, and proposed a specific region of protein C as a heparin-binding site.5. Modulation of Protein C Inhibitor Activity by Histidine-Rich Glycoprotein (HRG). We have found that HRG neutralizes the heparin-dependent inhibitions of activated protein C and thrombin by protein C inhibitor at physioloical concentrations of zinc and calcium ions in plasma, suggesting a new function of HRG as a physiological modulator of protein C inhibitor.6. Assignment of the Gene for HRG to Chromosome 3. We have assigned the gene for HRG to chromosome 3.